Understanding how information is processed in the brain would benefit from precise spatio-temporal recording of electrical activity in individual neurons and larger neuronal circuits. Optical reporting of neuronal activity using genetically encoded fluorescent reporters is a promising approach, as it allows for a set of neurons in a region to be genetically defined and simultaneously visualized without the need for chemical access. To unravel the rules of neural coding, an ideal sensor should not only be able to detect all types of membrane electrical activity—not only single action potentials (APs), or spikes, that represent neuronal outputs, but also monitor the amplitude and spatio-temporal distributions of inputs that are received and integrated by neurons (Magee (2000) Nat. Rev. Neurosci. 1:181-190; Zecevic et al. (2003) Imaging nervous system activity with voltage-sensitive dyes, Curr. Protoc. Neurosci Chapter 6, Unit 6.17). The ability to visualize subthreshold potential changes would also allow tracking of up and down states throughout a population of neurons, enabling questions regarding synchrony to be addressed (Castro-Alamancos (2009) Neuroscientist 15:625-634). Finally, tracking high-frequency firing would be useful for visualizing how bursts of neurotransmission are decoded by the postsynaptic neuron (Branco & Hausser (2011) Neuron 69:885-892) and for understanding how the 50-200 Hz firing of fast-spiking interneurons regulates information processing in the brain (Puig et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105:8428-8433; Royer et al. (2012) Nat. Neurosci. 15:769-775) or is affected in disease (Zhou & Roper (2011) Cereb. Cortex 21:1645-1658).
A genetically encoded sensor with these multiple capabilities remains to be developed. Following intense engineering efforts, reporters for calcium based on fluorescent proteins can now detect single action potentials (Chen et al. (2013) Nature 499, 295-300); however, they remain unable to monitor subthreshold depolarizations and hyperpolarizations, since those events are not typically characterized by large calcium fluxes. Furthermore, calcium transients persist in neurons for hundreds of milliseconds, and thus calcium responses cannot track high-frequency action potential trains (Murthy et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:901-906). For example, GCaMP6f, the fastest variant of the latest iteration of calcium sensors, has a mean decay time (τ1/2) of 400 milliseconds (Chen et al. (2013) Nature 499:295-300). Voltage sensors provide a more direct measure of membrane potential and, in some cases, are faster than calcium sensors. Sensors based on seven-helix microbial rhodopsin are a promising new class of voltage sensor, but currently cannot report neuronal activity over background fluorescence in brain slices or in vivo (Kralj et al. (2012) Nat. Methods 9:90-95; Gong et al. (2013) PLoS One 8:e66959). Moreover, the 5- to 15-milliseconds on- and off-rates of Arch-EEQ (Gong et al., supra), the fastest rhodopsin-based non-conducting sensor to date, are still slow compared to the typical 2-milliseconds duration of action potentials in pyramidal neurons (Staff et al. (2000) J. Neurophysiol. 84:2398-2408). Voltage sensors based on four-helix transmembrane voltage-sensing domains (VSDs) require light at the more phototoxic <450 nm CFP-exciting wavelengths (Lundby et al. (2008) PLoS One 3:e2514), produce suboptimal fluorescence responses to neuronal activity (Lundby et al., supra), or exhibit inactivation kinetics substantially slower than needed to follow fast trains of action potentials (Staff et al., supra). In particular, the recently developed ArcLight family of sensors produce the largest fluorescence response to APs among previously reported VSD-based sensors and are relatively bright, but slow kinetics limit their ability to resolve spikes separated by less than about 50 milliseconds (Cao et al. (2013) Cell 154:904-913; Jin et al. (2012) Neuron 75:779-785). Thus, no existing genetically encoded activity sensor possesses all the characteristics needed for accurate optical reporting of neuronal activity in vivo.
Thus, there remains a need for better voltage sensors and methods for imaging neuronal electrical activity.